Method of recovering broteolytic pancreas enzymes from mammalian pancreas glands subsequent to insulin removal



United States Patent.

METHOD OF RECOVERING BRQTYEOLYTIC P-AN- CREAS ENZYMES FROM PAN- CREAS GLANDS SUBSEQUENT TO INSULIN REMOVAL SCl'aims; (Cli 1 67 -75) The present inventionrelat'es to a methodlin recovering theproteolytic pancreas enzymes, in the followingsimply called .enzymes, from mammals, particularly horned. cattle.

and pigs, in connection with the preparation of insulin from the same glands. i y

The technical recovery of insulin from pancreas glands of mammals, particularly horned cattle and pigs, is carried out by treating the minced glands with suitable extracting,

agentsable to dissolve theinsulin and'under'suchcircumstances that the enzymesare preventedfrom inactivating the insulin. In the literature water, ethyl and methyl alcoliol, inthe following simply called alcohol,- acetone or mixtures of these substances with or without addition of acids, alkalies or salts, are indicated as suitable extracting agents. However, his common tomost ofthe methods that alcohol 01'- acetone is added at a certain moment either at once, i. 'e. to: the' minced glandsthemselves or lateron to a for instance aqueous.extract-thereof and then the insulin may be prepared inpure form without particular regard being paid to the enzymes and without any traceable inactivation of the insulin taking place;

In the glands taken out of the animals; immediately after slaughtering no active enzymes are found; enzymes are present in a completely inactivecondition, but they will rapidly be transformed into the active condition unless the glands-are at once frozen down tobelow 0 C. As the active enzymes act inactivating upon the insulin, even at a low temperature; always fresh or wellpreserved glands, i. e. immediately frozen glands, inwhich the enzymes consequently are completely or almost completely inactive, are used in the preparation of insulin to obtain the best possible yields thereof;

When pure insulincan be produced by the extraction methods mentioned above without any traceable inactivation of the insulin taking place this has been attributed to the employed alcohol or acetone which, according to some investigators, destroys the enzymes. Gonsequently, the substances separated during the insulin preparation-have been considered as unsuitable for recovering the-enzymes of the pancreas glands and, generallyspeaking, thesaid substances have been considered as a residual material which may onlybe usedas fodder'etc.

Therefore, in the production of pancreas-enzyme preparations which have lately found ,varioustechnical and pharmaceutical applications it has hitherto been necessary to use the glands directly either by extracting the-enzymes by treatment with suitable extracting agents, preferably aqueous liquids, orby simply drying the glands, removing their fats, if desired, and pulverizing' them, such' glands beingused in which. all enzymes are activated by storage for a shorter orlonger. period'dependent on 'theztemperature and at any rate so long that allinactiveenzyme has been transformed into active enzyme.

The present invention is based upon the new observa- The tion that the enzymes are. not destroyed during the treatment with alcohol. or acetone for the purpose of recoveringinsulin, butthat-hereby a precipitation of the enzymes is. effectedcontingent upon the concentration of the employed alcohol. or acetone. However, the precipitation also depends on the. proportion of liquid. and substance during the extraction. (the ratio of extraction.) but if not quite extreme. proportions are: used, the precipitation will occur at. above 20 to 60% of. alcohol or acetone, 20% applying; to a. small ratio of. extraction and 60%. to a large ratio of extraction- Thus, the enzymes are in reality found in. the residuefrom: the. recovery of insulin. when 20 to 601% of. alcohol or "acetone are used for the. recovery. Among the numeroussubstances separated. during'the purificationof. the insulin noenzyme is found. Thus, the good result of the. insulin recovery is obviously caused'by the fact. thatat a. given momenta solvent is used which under theconditions in. question at. the-same time acts as a precipitatingagentfor.theenzymes. The latter are consequently present in undissolved. or inactive condition .in the residue. fromthepreparation of theinsulin from fresh or well-preservedglands'andcannot be activated until they are resolved.

In. order tov obtain an. active enzyme preparationfrom the residue from the insulinrecovery. one should conse quently, according. to the: invention, only take care of .dissolving. theprecipitated. enzymes.- and": then. subjecting themto activation.

1m accordance. herewith the method accordingto the invention ischaracterized in.that the residue" arising dur-ing the. production. of. insulin. from minced fresh or wellpreserved glands by treatingrtherlatter or extractsthereof at. a suitable pHwalue. with: an amountvof alcohol or acetone sufficient to produce an alcohol or acetone concentration above.20 to.60.% and separatingfihe liquid, is subjectedto ancnzyme extractionin thepresence of: below 20. to 60% of alcohol or acetone. Expressedinanother way, during. the: insulin preparation the concentration of alcohol. or. acetone. should be above 30: 10%, while an alcohol oracetone concentrationsbelow 3'0: 10% should be used duringtthe. extraction of the enzymes, the

value 30+-1.0% (i. e. 4.0% being used. inconnection with a large ratio of extraction and. the. value 30-10% (i. e. 20% in connection with asmallratioof extraction.

According/to the presentinvention'it willconsequently glands up to a concentration oflil to 60%,.working up the.insulin.from,this liquid and themsubjectingthe.residue to an enzyme extraction, care being taken that: below .20.to 60% of alcohol or acetone are present. during the saidextraction, or in. other words substituting the precipitating agent by an enzymedissolving agent. and then activating the dissolved enzyme...

For further illustratingthe. importanceof the. alcohol or acetone concentration it. shall. be. mentioned, ethyl alcohol being used as an. example, that with a: ratio of extraction of 7:1. the precipitation. of. the enzymes be gins at about 30% alcohoLand. is practically quantitative at about 40%. Consequently, in.this case antethyl alcoliol concentration of above 40%. oughtxto. beused during the extraction of the insulin while the alcohol concentration ought to lie below- 30% during the enzyme extraction.

The yieldof. enzymes. obtainable: by the method. on the invention dependson. the conditions. during. the insulin recovery and during, the. enzyme. recovery;

As toathe conditions duringthe. insulin recovery the yield of enzymes is independent of the employed alcohol Patented Aug. 20, 1957 p using 7 parts of the extracting liquid. As it appears from the above stated it is decisive for or acetone concentration when the latter is above 20 to 60% and independent of the employed period of extraction if only the latter has been so long that the employed alcohol or acetone has come into contact with the enzymes and precipitated them. As to the conditions of the enzyme recovery the yield primarily depends on the concentration of the alcohol or acetone used. At 20 to 60% alcohol or acetone and higher-dependent on the ratio of extractionthe enzymes remain undissolved, and below the said concentration the enzymes begin to dissolve and the yield'is then highly increasing with decreasing alcohol or acetone concentration. Also the extraction period plays a role so that reduced extraction periods give decreasing yields. Technically it is preferred to use Water for dissolving the enzymes and it has been found that an extraction with water for a period of 24 hours gives good yields when the yield of enzymes that the contents of alcohol or acetone in the residue from the insulin manufacture are brought down below 20 to 60%. To obtain this a residue with high alcohol or acetone contents may be treated with sufiicient amounts of the enzyme dissolving agent, for instance water. It is more appropriate first to remove the main portion of the alcohol or acetone contents of the residue, for instance by pressing, and then to effect the enzyme extraction. Hereby more concentrated enzyme solutions and better conditions for recovering the employed alcohol or acetone, are obtained.

The method according to the invention particularly aims at a utilization of the gland residue fro-m the acid alcoholic insulin extraction. When for instance hydrochloride acid, phosphoric acid, lactic acid, or acetic acid is used during the said extraction the enzymes may be extracted from the residue by means of water when care is taken that below 20 to 60% alcohol are present during the extraction.

On the other side, when sulphuric acid is used in extracting the insulin it has been found that the enzymes are present in the residue in such a condition that they cannot be extracted directly. even if the alcohol or acetone concentration is zero during the enzyme extraction, the extraction period is long and the amount of extracting liquid is large. However. according to the invention an extraction is successful when a strong solution of urea, above 50% acetic acid, 85% formic acid. liquid phenol is used as extracting agent or when a cautious extraction by means of alkaline medium is employed. According to the invention it has been found, however. that a simple aoueous extraction may also be used if the residue is subjected to a short heating, for instance at 100 C.

As to the pH-value during the enzyme extraction it ought to lie between about 1 and 7, preferablv 1 and 4.

' Finally it may be remarked that my Patent No. 2,524,- 658, dated October 3, 1950 (Ser. No. 705,247 filed October 23, 1946) relates to a method of simultaneously recovering enzymes and insulin from pancreas, in which insulin and enzymes may also be recovered from the same gland material. However, this method is principally diiferent from the method of the invention since it consists in first extracting the enzymes from fresh or well-preserved glands by means of an acid aqueous ex-' tracting agent at low temperature and/ or with short period of extraction and then recovering the insulin from the extraction residue according to known methods.

The following examples illustrate the method according to the invention:

is minced and extracted for 3 hours with 4 litres of 80% ethyl alcohol to which 30 cc. of concentrated hydrochloric acid have been added. The liquid is filtered ofi ately thereafter.

and may be worked up into insulin in the usual manner while the residue is subjected to enzyme extraction for 24 hours with 7 litres of water and then centrifugated and the enzymes are recovered from the liquid. A yield of about 10 million Fuld-Gross units is obtained.

Example 2 Example 3 l kilogram of fresh or Well-preserved pancreas glands is minced and extracted for 3 hours with 2 litres of ethyl alcohol to which 10 cc. of concentrated sulphuric acid have been added. The liquid is centrifugated and may be worked upinto insulin in the usual manner while the residue is subjected to enzyme extraction with 3 litres of 33% urea solution for 2 hours.

After centrifugation the enzymes are recovered from the liquid. A yield of about 8 million Fuld-Gross units is obtained.

Example 4 1 kilogram of fresh or well-preserved pancreas glands is mincedand extracted for 8 hours with 6 litres of 80% .fethyl alcohol to which are added 10 cc. of concentrated sulphuric acid. The liquid is centrifugated and may be worked up into insulin in the usual manner while the residue after pressing is mixed with 4 litres of water, to which 20 cc. of concentrated hydrochloric acid are added, and heated quickly to C. and cooled again immedi- After centrifugation the enzymes are recovered from the liquid. A yield of about 9 million Fuld-Gross units is obtained.

These yields express average yields. With particularly high class gland material considerably higher yields may be obtained.

This application is a division of my co-pending application Serial No. 705,248 filed October 23, 1946, now Patent 2,571,126.

Having thus fully described my invention I claim as new and desire to secure by Letters Patent:

1. A method of recovering proteolytic enzymes from mammalian pancreas glands, which comprises adding water to the residual gland material resulting from the treatment of mincedpancreas gland'with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 7, thereby bringing the proteolytic enzymes contained in the residual gland material into solution in an acid milieu, and recovering the dissolved enzymes.

2. A method of recovering proteolytic enzymes from mammalian pancreas glands, which comprises removing the main portion of the insulin extracting solution from the residual gland material resulting from the treatment of minced pancreas gland with a conventional insulin extracting concentration of a solvent selected from the 'group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the -group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 7, adding water to saidre'sidual gland material, thereby bringing the proteolytic enzymes contained in the residual gland .material into solution in an acid milieu, and recovering the dissolved enzymes.

3. A method in recovering insulin and proteolytic panci'eas enzymes from mammalian pancreas glands, which comprises treating a minced pancreas gland material with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 7, separating the insulin-containing liquid from the treated gland material, recovering the dissolved insulin from the separated liquid, adding water to the separated residual gland material to dissolve the proteolytic enzymes, and recovering the dissolved enzymes.

4. A method in recovering insulin and proteolytic pancreas enzymes from mammalian pancreas glands, which comprises treating a minced pancreas gland material with a conventional insulin extracting concentration of a solvent .selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 7, separating the insulin-containing liquid from the treated gland material, recovering the dissolved insulin from the separated liquid, removing the main portion of the insulin extracting solvent from the separated residual gland material, adding water to the residual gland material to dissolve the proteolytic enzymes, and recovering the dissolved enzymes.

5. A method of recovering proteolytic enzymes from mammalian pancreas glands, which comprises adding water to the residual gland material resulting from the treatment of minced pancreas gland with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 4, thereby bringing the proteolytic enzymes contained in the residual gland material into solution in an acid milieu, and recovering the dissolved enzymes.

7 6. A method of recovering proteolytic enzymes from mammalian pancreas glands, which comprises removing the main portion of the insulin extracting solution from the residual gland material resulting from the treatment of minced pancreas gland with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 4, adding water to said residual gland material to create an acid l 6 milieu of pH 1 to 4, thereby bringing the proteolytic enzymes contained in the residual gland material into solution in an acid milieu, and recovering the dissolved enzymes.

7. A method in recovering insulin and proteolytic pancreas enzymes from mammalian pancreas glands, which comprises treating a minced pancreas gland material with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 4, separating the insulin-com taining liquid from the treated gland material, recovering the dissolved insulin from the separated liquid, adding water to the separated residual gland material to dissolve the proteolytic enzymes, and recovering the dissolved enzymes.

8. A method in recovering insulin and proteolytic pancreas enzymes from mammalian pancreas glands, which comprises treating a minced pancreas gland material with a conventional insulin extracting concentration of a solvent selected from the group consisting of methyl alcohol, ethyl alcohol, and acetone acidified with a mineral acid selected from the group consisting of hydrochloric acid and phosphoric acid to create an acid milieu having a pH below 4, separating the insulin-containing liquid from the treated gland material, recovering the dissolved insulin from the separated liquid, removing the main portion of the insulin extracting solvent from the separated residual gland material, adding water to the residual gland material to create an acid milieu of pH 1 to 4 to dissolve the proteolytic enzymes, and recovering the dissolved enzymes.

References Cited in the file of this patent UNITED STATES PATENTS 412,836 Carnrich Oct. 15, 1889 1,469,994 Banting et a1. Oct. 9, 1923 2,353,016 Daughenbaugh July 4, 1944 2,524,658 Frederiksen Oct. 3, 1950 FOREIGN PATENTS 445,131 Belgium of 1942 OTHER REFERENCES Kunitz et al.; Science, Dec. 15, 1933 pp. 558-559. Rabinowitch et a1.: Biol. Chem, November 1938, pp. 109-115. 

1. A METHOD OF RECOVERING PROTEOLYTIC ENZYMES FROM MAMMALIAN PANCREASE GLANDS, WHICH COMPRISES ADDING WATER TO THE RESIDUAL GLAND MATERIAL RESULTING FROM THE TREATMENT OF MINCED PANCREAS GLAND WITH A CONVENTIONAL INSULIN EXTRACTING CONCENTRATION OF A SOLVENT SELECTED FROM THE GROUP CONSISTING OF METHYL ALCOHOL, ETHYL ALCOHOL, AND ACETONE ACIDIFIED WITH A MINERAL ACID SELECTED FROM THE GROUP CONSISTING OF HYDROCHLORIC ACID AND PHOSPHORIC ACID CREATE AN ACID MILLIEU HAVING A PH BELOW 7, THEREBY BRINGING THE PROTEOLYTIC ENZYMES CONTAINED IN THE RESIDUAL GLAND MATERIAL INTO SOLUTION IN AN ACID MILLIEU, AND RECOVERING THE DISSOLVED ENZYMES. 